Method for determining activity of cell cycle regulatory factor and method for diagnosing cancer using the same

ABSTRACT

A method for determining the activity of a cell cycle regulatory factor comprising the steps of preparing a sample for measuring a cyclin-dependent kinase/cyclin complex from living cells; reacting adenosine 5′-O-( 3 -thiotriphosphate) (ATP-γ S) with a substrate for the cyclin-dependent kinase in presence of the sample in order to introduce a monothiophosphate group into a serine or threonine residue of the substrate; labeling the substrate by coupling a labeling fluorophore or a labeling enzyme with a sulfur atom of the introduced monothiophosphate group; measuring the amount of fluorescence from the labeling fluorophore labeling the substrate, or reacting the labeling enzyme labeling the substrate with a substance which generates an optically detectable product by reaction with the labeling enzyme and optically measuring the amount of the generated product; and calculating the activity of the cyclin-dependent kinase from the measured amount of fluorescence or the measured amount of the generated product with reference to a pre-produced reference curve.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 37 C.F.R. § 1.53(b) divisional application of U.S.application Ser. No. 10/074,041 filed Feb. 14, 2002, which claimspriority under 35 USC § 119 of Japanese Patent Application No.2001-37115 filed on Feb. 14, 2001. Each of these applications isincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for determining the activityof a cell cycle regulatory factor without using a radioisotope and amethod for diagnosing a cancer using the method.

2. Description of Related Art

Cell proliferation is a fundamental and important feature of livingthings. The cell proliferation involves division of a single cell intotwo daughter cells, and somatic cells divide through a plurality ofsequential reactions including growth of cells, replication of DNAs,distribution of chromosomes and division of cells. This chain ofsequential reactions is referred to as a cell cycle. In the case ofeucaryotic cells, the cell cycle is divided into four phases, that is, asynthetic (S) phase during which the replication of DNAs takes place, amitotic (M) phase during which the division of cells takes place, a gap1 (G1) phase which is an interphase from the M phase to the next S phaseand a gap 2 (G2) phase which is an interphase from the S phase to thenext M phase. In the G1 phase, cells receive a signal for proliferation,prepare for the replication of DNAs, and make metabolism and growthwhich are necessary for the division of cells. In the G2 phase, thecells prepare for the division. In the G1 phase, a transit point isexperimentally assumed which is called an R point (restriction point)for mammalian cells and START for yeast. Typically, cells multiply inresponse to proliferation signals from the outside. The cells receivethe signals in the G1 phase and progress the cell cycle. After passingthrough a certain point in the G1 phase, the cell cycle progresses fromthe S phase to the G2 phase, the M phase and then the G1 phase withoutstopping even if the proliferation signals are not received any longer.This certain point is the R point or START, and is a so-to-speak pointat which the entering into cell cycle progression is determined.Further, the cells can leave the cell cycle and enter a resting (G0)phase during which the cell do not grow or multiply. Experimentally, thecells entering the resting phase, if given a suitable signal, can bereturned to the G1 phase and induced to grow and divide again. It isconsidered that a lot of non-growing and non-multiplying cells ofmulticellular organisms are in the G0 phase.

There mainly exist two groups of cell cycle regulatory factors in cells.One is a group of kinases which are positive regulatory factors and arereferred to as cyclin-dependent kinases (CDKs), and the other is a groupof CDK inhibitors (CDKIs) which are negative regulatory factors. TheCDKs exist in cytoplasm in the inactive form. The CDKs are activated,e.g., by phosphorylation, and move into nuclei in the cells. In thenuclei, the CDKs bind to cyclin molecules to form complexes with cyclin(referred to as activated CDKs hereinafter) and positively regulate theprogress of the cell cycle at various steps of the cell cycle. On theother hand, the CDKIs inactivate the CDKs by binding to the activatedCDKs or CDK simple substances, thereby regulating the cell cyclenegatively.

There are now known seven types of CDKs, i.e., CDK1, CDK2, CDK3, CDK4,CDK5, CDK6 and CDK7 to which different cyclins are bound. Moreparticularly CDK1 binds to cyclin A or B, CDK2 binds to cyclin A or E,and CDK4 and CDK6 bind to cyclin D1, D2 or D3, to be activated. Theactivated CDKs control specific phases of the cell cycle. The followingtable 1 shows CDKs concerning the control of the cell cycle, cyclinswhich functionally bind to the CDKs, and phases of the cell cycle duringwhich the activated CDKs act. TABLE 1 Cyclins binding to Phases of cellcycle in CDKs CDKs which activated CDKs act CDK4, CDK6 Cyclin D1, D2, D3G1 CDK2 Cyclin E Transitional period from G1 to S CDK2 Cyclin A S CDK1Cyclin A, B Transitional period from S to M, M

Thus the cell cycle is controlled and the cell proliferation isregulated by activation of different types of CDKs. The activated CDKsare enzymes which phosphorylate serine residue and threonine residue ina protein as a substrate. In an in-vitro reaction system, the activatedCDK1 and CDK2 react well on histone H1 as a substrate and the activatedCDK 4 and CDK6 react well on Rb (retinoblastoma protein) as a substrate.In an in-vivo cell cycle regulation, it is considered that the activatedCDKs require Rb as a physiologic substrate, but it is not known whatother proteins act as substrates.

As described above, the CDKs and cyclins regulate the cell cycle inclose association with each other. The multiplication of cyclin D1 geneis observed in a great number of cases of esophageal cancer, while overexpression of cyclin D1 gene is observed in a great number of cases ofstomach cancer and colon cancer. On the other hand, the multiplicationof cyclin E gene is observed in stomach cancer and colon cancer but isnot observed in esophageal cancer. Excessive expression of cyclin E instomach and large bowel takes place with great frequency in cases ofadenoma and adenocarcinoma and shows a significant correlation withmalignancy such as invasion, progress of stages, metastasis and thelike. The expression and kinase activity of CDK1 are remarkablyaccelerated in most cases of stomach cancer and colon cancer as comparedwith normal mucosal tissue. It is known that augmented expression ofcyclin genes correlates with the progress and malignancy of variouscancers (see Wataru Yasui, Sysmex Journal Web., p. 1 to p. 10, vol. 1,2000).

Therefore, it is expected that measuring the activity of the individualspecies of CDKs will provide good indices of the type and malignancy ofdiseases related to the control of the cell cycle. In other words,generally at the R point, the expression of CDK2 decreases and the cellcycle arrest and the division of cells is controlled. However, if theexpression of CDK2 increases at the R point, it means that the cellcycle fails to stop, i.e., it means a state of a disease such as cancer.If the expression of CDK4 or CDK6 increases, stomach cancer or coloncancer may be expected because stomach cancer and colon cancer involveaccelerated gene expression of the cyclin D1 which bind specifically toCDK4 or CDK6. Thus, it is considered to be possible to determine thetype of cancer.

Usually, the activity of the CDKs is determined using radioisotopes.More particularly, in the presence of a CDK which is extracted from acell lysate by an immunoprecipitation method using an anti-CDK antibodyand whose activity is unknown, ³²P-labelled adenosine5′-O-(3-triphosphate) (ATP) is reacted with serine residue or threonineresidue in a substrate to introduce monophosphate group derived from the³²P-labeled ATP. The amount of ³²P taken by the substrate is detected byautoradiography or by a scintillation counter. Thereby the amount of thephosphorylated substrate is measured and the activity of the CDK iscalculated from the amount of the phosphorylated substrate.

This method requires careful attention in handling the substance and indisposal of waste liquid since it uses ³²P which is a radioisotope.

SUMMARY OF THE INVENTION

Accordingly, are desired a method for measuring cell cycle regulatoryfactors accurately without using radioisotopes and a method fordiagnosing cancer on the basis of measurement results.

The present invention is to provide a method for determining theactivity of a cell cycle regulatory factor comprising the steps of:

preparing a sample for measuring a cyclin-dependent kinase/cyclincomplex from cells;

reacting adenosine 5′-O-(3-thiotriphosphate) (ATP-γ S) with a substratefor the cyclin-dependent kinase in presence of the sample in order tointroduce a monothiophosphate group into a serine or threonine residueof the substrate;

labeling the substrate by coupling a labeling fluorophore or a labelingenzyme with a sulfur atom of the introduced monothiophosphate group;

measuring the amount of fluorescence from the labeling fluorophorelabeling the substrate, or reacting the labeling enzyme labeling thesubstrate with a substance which generates an optically detectableproduct by reaction with the labeling enzyme and optically measuring theamount of the generated product; and

calculating the activity of the cyclin-dependent kinase from themeasuring amount of fluorescence or the measured amount of the generatedproduct with reference to a pre-produced reference curve.

Further, the present invention is to provide a method of diagnosing acancer based on a result obtained by determination.

These and other objects of the present application will become morereadily apparent from the detailed description given hereinafter.However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reference curve representing a relation between theamount (ng/slot) of biotinated actin and the amount of fluorescence(count) obtained in Example 1;

FIG. 2 shows a reference curve representing a relation between theamount (ng/slot) of biotinated actin and the amount of fluorescence(count) obtained in Example 2;

FIG. 3 shows a fluorescent band obtained in Example 3;

FIG. 4 shows a reference curve for measuring the activated CDK2;

FIG. 5 shows a fluorescent band obtained in Example 5; and

FIG. 6 is a graphical representation obtained in Example 6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For carrying out the method of the present invention, a sample is firstprepared.

In the present invention, the sample which contains a cyclin-dependentkinase (CDK)/cyclin complex may contain a single type or plural types ofCDK/cyclin complexes, but may preferably contain a single type ofCDK/cyclin complex.

The sample which contains a CDK/cyclin complex (referred to as anactivated CDK hereinafter) and is used in the method of the presentinvention is prepared by solubilizing cells and separating a samplecontaining the activated CDK to be determined from a liquid containingthe solubilized cells.

CDKs usable in the present invention include CDK1, CDK2, CDK3, CDK4,CDK5, CDK6 and CDK7.

(1) Step of Solubilizing Cells

The sample is prepared from cells derived from animals including humanbeings such as a tissue sample (e.g., a biopsy sample, a surgicallyresected sample, etc.). The sample is to be tested as to whether or notit contains a CDK/cyclin complex as well as its activity. Since simpleCDKs exist in cytoplasm and turn into activated CDKs by binding tocyclins in nuclei, cells need to be solubilized to extract the activatedCDKs.

Accordingly, the cells are first solubilized by chemically or physicallydestroying the cell membranes and nuclear envelopes thereof. Moreparticularly, the cells are preferably pulverized using a Waringblender, sucked and discharged using a syringe or ultrasonicated in abuffer containing a surfactant, a protease inhibitor and a phosphataseinhibitor, for example.

The surfactant is used for destroying cell membranes and nuclearenvelopes so that intracellular substances can be taken out. However,the surfactant should have such a surface active property that theactivated CDK is not decomposed. Examples thereof include Nonidet P-40,Triton X-100, deoxycholic acid and CHAPS. The concentration of thesurfactant is preferably 1 w/v % or less.

The protease inhibitor is used for preventing the CDK and cyclinmolecule, which are proteins, from being destroyed when mixed withintracellular substances when the cell membranes and nuclear envelopesare destroyed. Examples thereof include a mixture of a metalloproteaseinhibitor such as EDTA, EGTA, etc., a serine protease inhibitor such asPMSF, trypsin inhibitor, chymotrypsin, etc., and/or a cysteine proteaseinhibitor such as iodoacetaminde, E-64, etc., and a protease inhibitorcocktail commercially available from Sigma which contains such proteaseinhibitors premixed.

The phosphotase inhibitor is used for preventing the activated CDK,which is protein itself, from changing its activity by hydrolysis of itsphosphate group. Examples thereof include a serine/threonine phosphotaseinhibitor such as sodium fluoride and a tyrosine phosphotase inhibitorsuch as sodium orthovanadate (Na₃VO₄).

After the cells are solubilized, insoluble matters are removed from celllysate by centrifugation or by filtration using a filter. Subsequently,prior to separating the sample containing the activated CDK, it ispreferable to measure the total amount of proteins in the cell lysateaccording to a method known to those skilled in the art. Example, thetotal amount of proteins may be measured using a DC protein kit usingbovine IgG as a reference.

(2) Separation of Sample Containing Activated CDK to Be Determined

The sample containing the activated CDK whose activity is to bedetermined is prepared from the thus obtained cell lysate. The samplecontaining the activated CDK can be prepared, for example, by animmunoprecipitation method.

According to the immunoprecipitation method, is used an anti-CDKantibody having a specificity to one of the CDKs 1 to 7 to bedetermined.

More particularly, the cell lysate containing a specific amount ofprotein is reacted with an anti-CDK antibody corresponding to theactivated CDK to be determined and a suspension of sepharose beads (abeads content of 4 and 6 v/v %) coated with Protein A, Protein G oranti-rabbit IgG antibody as material for catching the anti-CDK antibodyat 0 to 10° C. for one to two hours. Since these beads are insoluble,the complex of the anti-CDK antibody and the CDK bound to the beadsbecome insoluble and precipitate.

By immunoprecipitation, all CDK family (including the simple CDK, theactivated CDK, the complex of the activated CDK and CDKI and the complexof the CDK and CDKI) in the liquid containing solubilized cells arecaught. Accordingly, the activated CDK are contained together with thesimple CDK, the complex of the activated CDK and CDKI and the complex ofCDK and CDKI in the prepared sample. However, the inactivated CDK doesnot involve monothiophosphorylation of the substrate in the presence ofATP-γ S. If the present invention is carried out on a sample containingthe activated CDK for determining the activity of the activated CDK, theactivity of the inactivated CDK is not detected and only the activity ofthe activated CDK is determined.

Subsequently, the precipitated beads to which the complex of theactivated CDK and the anti-CDK antibody are bound are washed. A buffersolution for washing the beads contains magnesium chloride since theactivated CDK needs to form a complex with magnesium in order that ATP-γS acts on the substrate and the activated CDK later. The buffer solutionalso contains, for example, dithiothreitol (DTT) as a stabilizernecessary for stabilizing the molecular structure of the substrate.Further the buffer solution may contain albumin, a trace of a surfactantand/or the like.

Thereafter, the activity of the activated CDK in the sample isdetermined. The method of the present invention includesmonothiophosphorylating the serine or threonine residue of the substratein the presence of the activated CDK, labeling the resultingthiophosphorate group and measuring the label. In the present invention,the activated CDK bound to the CDK antibody caught by the beads may beused as an activated CDK.

(i) In the presence of the activated CDK, a substrate, which is asubstrate for the CDK, is reacted with adenosine5′-O-(3-thiotriphosphate (ATP-γ S) to introduce a monothiophosphategroup derived from ATP-γ S into the serine group and threonine group ofthe substrate.

Usually, activated CDKs act to react ATP with the serine or threoninegroup of the substrate to introduce a monophosphate group derived fromATP. However, in the present invention, ATP-γ S is used instead of ATPto introduce the monothiophosphate group instead of the monophosphategroup in the serine or threonine group of the substrate.

For monothiophosphorylation, a liquid of pH 6.5 to 8.5, preferably 7.4,containing 0.1 to 1.0 mg/mL of the substrate is reacted with 10 to 100equivalents of ATP-γ S with respect to 1 equivalent of the substrate inthe presence of the activated CDK at 25 to 40° C., preferably 37° C.,for 5 minutes to 1 hour, preferably 10 minutes.

As discussed above, the sample contains not only the activated CDK butalso the inactivated CDK. However, since only the activated CDKcatalyzes the thiophosphate group introduction reaction, the inactivatedCDK does not participate in the method of the present invention.

As examples of the substrate, histone H1 and Rb (Retinoblastoma protein)may be mentioned for the activated CDK1 and CDK2 and for the activatedCD4 and CDK6, respectively.

In the present invention, regarding substrates such as Rb whichessentially contain the cysteine residue in their molecules, the residueis substituted by an amino acid residue such as alanine which does notcontain thiol group. This is for avoiding measurement errors owing tothe labeling of the thiol group of the cysteine residue essentiallypresent in the substrate at the same time when sulfur atom ofthiophosphate group of the substrate (into which thiophosphate groupderived from ATP-γ S by the action of the activated CDK) is labeled withthe labeling fluorophore or the labeling enzyme.

Regarding a substrate which essentially contains the cysteine residue inits molecules, it may be possible to produce, from the substrate, asubstrate of which the cysteine residue is substituted by an amino acidresidue such as alanine which does not contain the thiol residue, by PCRor by modifying a gene of the substrate by site mutagenesis andexpressing the modified gene. Particularly, with regard to a substratesuch as Rb which contains the cysteine residue, a recombinant vector isobtained by cloning with use of oligonucleotide primers Rb-1 (5′-ACA GGATCC TTG CAG TAT GCT TCC-3′), Rb-2 (5′-GCT GGT AGC TAC CAT CTG ATTTAT-3′), Rb3 (5′-ATG GTA GCT AAC AGC GAC CGT GTG-3′), and Rb-7 (5′-GCGAAT TCA ATC CAT GCT ATC ATT-3′); the recombinant vector is expressed toobtain a recombinant DNA in which a nucleotide coding cysteine residueis substituted by a nucleotide coding alanine residue; and therecombinant DNA is expressed to produce a substrate in which thecysteine residue is substituted by the alanine residue.

(ii) Labeling of the Thiophosphate Group Introduced in the Substrate andMeasurement of the Amount of the Label

In order to label the substrate by coupling the labeling fluorophore orthe labeling enzyme with sulfur atom of the introduced thiophosphategroup, a liquid of pH 7.5 to 9.0, preferably 8.5, containing 0.1 to 1.0mg/mL of the substrate into which the thiophosphate group is introducedis reacted with 10 to 100 equivalents of a labeling fluorophore or alabeling enzyme having a functional group which reacts with the thiolgroup, with respect to 1 equivalent of the substrate for 10 minutes to 2hours. This reaction is stopped by adding a free thio, for example, β-ME(β-mercaptoethanol), DTT (dithiothreitol) or the like.

In the case where the substrate is labeled with the labelingfluorophore, the amount of fluorescence from the labeling fluorophore ismeasured. The measured amount of fluorescence is compared with areference curve which has been produced beforehand from the amount offluorescence measured about the known amount of the substrate, andthereby the amount of the labeled substrate is calculated. The amount ofthe labeled substrate is regarded as an activity value of the activatedCDK contained in the sample.

In the case where the substrate is labeled with the labeling enzyme, thelabeling enzyme is reacted with a substance which generates an opticallydetectable substance by reaction with the labeling enzyme. The amount ofthe generated product is optically measured and the measured amount iscompared with a reference curve which has been produced beforehand, andthereby the activity value of the activated CDK contained in the sampleis calculated. Here the optically detectable substance means a substancewhose existence can be detected by measuring fluorescence, absorbanceand/or the like of the substance.

As examples of the labeling fluorophore capable of binding to the sulfuratom of the thiophosphate group, may be mentioned fluorescein, coumarin,eosin, phenanthroline, pyrene, Rhodamine and the like, among whichfluorescein is preferred. In order that the labeling fluorophores bindsto the sulfur atom of the thiophosphate group, the labeling fluorophoreshave functional groups such as an alkyl halide, maleimide, aziridinesite and the like which react with the thio group for labeling thesulfur atom of the thiophosphate group.

As examples of the labeling fluorophore having a functional group whichreacts with the thiol group, may be mentioned iodoacetyl-FITC(fluorecein isothiocyanate), 5-(bromomethyl)fluorecein,fluorecein-5-maleimide, 5-iodoacetamidefluorecein (5-IAF),6-iodoacetamidefluorecein (6-IAF), 4-bromomethyl-7-methoxycoumarin,eosin-5-iodoactamide, eosin-5-maleimide, eosin-5-iodoacetamide,N-(1,10-phenenthrolin-5-yl)bromoacetamide, 1-pyrenebutylchloride,N-(1-pyreneethyl)iodoacetamide, N-(1-pyrenemethyl)iodoacetamide (PMIAamide), 1-pyrenemethyliodoacetate (PMIA ester), Rhodamine red C2maleimide and the like, among which iodoacetyl-FITC is preferable.

Alternatively, the labeling fluorophore may be introduced tothiophosphate group by reacting the molecule with biotin which has afunctional group reacting with sulfur atom of thiophosphate group, forexample, iodoacetylbiotin, and then reacting the molecule with alabeling fluorophore covalent-bound to avidin for taking advantage ofthe affinity of biotin to avidin.

The labeling enzyme may be introduced to sulfur atom of thiophosphategroup by introducing iodoacetylbiotin to sulfur atom and then reactingthe molecule with a labeling enzyme covalent-bound to avidin which hasaffinity to biotin. As such enzymes, may be mentioned β galactosidase,alkaline phosphatase, peroxidase and the like, among which peroxidase ispreferred.

The amount of the labeled substrate may be measured by measuring theamount of fluorescence from the labeling fluorophore or by allowing asubstance which generates an optically detectable product by reactionwith the labeling enzyme to act on the substrate labeled with thelabeling enzyme and then optically measuring the generated product.

More particularly, where the labeling fluorophore is used, the labelingfluorophore is excited by a specific wavelength and analyzed by afluorescent image analyzer. The wavelength of applied light may varydepending upon the type of a labeling fluorophore used. For example,light of 488 nm wavelength is applied for excitation where the labelingfluorophore is fluorescein.

Where the labeling enzyme is used, a substrate which will produce afluorophore by reaction with the labeling enzyme is added to thesubstrate labeled with the labeling enzyme in order to produce thefluorophore by reaction with the labeling enzyme. The producedfluorophore is excited by light having a specific wavelength and theemitted fluorescence is detected. The substrate which produces afluorophore by reaction with the labeling enzyme may be ECL-plus in thecase where the labeling enzyme is peroxidase. The substrate may beselected as appropriate depending upon a labeling enzyme used.

The amount of the labeling fluorophore or the amount of the fluorophoreproduced by the reaction is measured and applied to the reference curvemade beforehand in order that the activity of the activated CDK iscalculated. The reaction liquid of the labeled substrate needs to bediluted to such a degree that the amount of fluorescence from thelabeling fluorophore or the fluorophore produced by the reaction withthe labeling enzyme falls within the range of the reference curve. Forexample, the reaction liquid may be diluted 100 to 500 fold. Asdiluents, may be used TBS (50 mM Tris-HCl of pH 7.5, 150 mM NaCl),water, an aqueous sodium chloride solution and the like. In the case ofaqueous sodium chloride solution, the concentration of sodium chloridemay preferably be in the range of 100 to 500 mM. In the case where thereaction liquid is diluted, the dilution is taken into account when theactivity of the activated CDK is the activity of the activated CDK in aspecific amount of protein taken from the total protein of the preparedsample.

The reference curve is preferably produced beforehand using a knownamount of the substrate to which thiol group is introduced. Alsobiotinylated actin may be used instead. Biotinylated actin is known tohave the same behavior to reaction with the labeling fluorophore and thelabeling enzyme as the substrate to which thiol groups has beenintroduced. In this case, the activity of the activated CDK is requiredto be calculated from the amount of biotinylated actin.

The present invention also provides a method for diagnosing cancers suchas stomach cancer, colon cancer, breast cancer, lung cancer, esophagealcancer, prostate cancer, hepatic cancer, kidney cancer, bladder cancer,skin cancer, uterine cancer, cerebral tumor, osteosarcoma and myeloma,from the results of the CDK activity determined by the determinationmethod of the present invention.

EXAMPLES

Exemplary Method 1 (Determination of Activated CDK1: Using Histone H1 asa Substrate and Utilizing Peroxidase-Labeling)

First Step:

In ice bath, HeLa cells (carcinoma cells of uterine cervix) were lysedin a lysis buffer containing 0.1 w/v % NP40 (surfactant Nonidet P-40),50 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM sodium fluoride, 1 mM sodiumorthovanadate and 100 μL/mL protease inhibitor cocktail (Sigma), in aproportion of 1×10⁷ cells/5 mL buffer, by 10 times repeated sucking anddischarging with a 5-mL syrine provided with a 23G needle. A cell lysatewas thus prepared. Insolubles were removed by centrifugation at 4° C. at15,000 rpm for 5 minutes. The total amount of protein contained in thesupernatant was measured by a DC protein kit (Bio-Rad) using bovine IgGas reference.

Second Step:

A sample was prepared by adding 10 μg of the lysed protein in the totalamount to 500 μL of the lysis buffer in an Eppendorf tube of 1.5 mLvolume. To the prepared sample, 10 μL of polyclonal anti-CDK antibody(Santa Cruz Biotechnology) were added. A 1:1 (sepharose beads:lysisbuffer) slurry of 60 μL sepharose beads (finally 30 μL of enclosedbeads) coated with Protein A was added to the resulting sample, whichwas incubated at 4° C. for an hour with continuous rotation. The beadswere taken out of the sample and washed with 1 mL of the lysis buffertwice. Then the beads were washed once with 1 mL of a kinase buffercontaining 50 mM Tris-HCl, pH 7.4, 10 mM magnesium chloride (MgCl₂) and1 mM DTT. The beads were suspended again in 15 μL of the kinase buffer.

Third Step:

To the obtained suspension, 10 μL of a histone H1 solution (0.1 mg/mLsolution in 50 mM Tris-HCl, pH 7.4) were added. Then 10 μL of an ATP-γ Ssolution (10 mM aqueous solution) were added to the suspension. Theresulting suspension was incubated at 37° C. for 10 minutes withcontinuous oscillation. The beads were precipitated by centrifugation at10,000 rpm for 10 seconds and 30 μL of supernatant were collected. Tothe supernatant, were added 25 μL of a binding buffer containing 150 mMTris-HCl and 5 mM EDTA of pH 9.2 since pH 8.5 is the optimum conditionfor binding reaction of iodoacetylbiotin with thiophosphoric acid. Tothe resulting supernatant, 20 μL of a 40 mM PEO-iodoacetylbiotin(Pierce) solution (in a 20 mM phosphate buffer of pH 6.0) were added.The supernatant was incubated in a dark place at room temperature for 90minutes. The reaction with iodoacetylbiotin was stopped by adding 7.5 μLof β-ME (β-mercaptoethanol). the reaction liquid was diluted with TBS(50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride).

Fourth Step:

The diluted reaction liquid, 50 μL, was placed and absorbed onto a PVDFmembrane using a slot blotter. The membrane was washed once with 50 mLof TBS-T (a TBS solution containing 0.05 w/v % Tween 20). In order toprevent the membrane to react with avidin-perioxidase, a hydrophobicpart of the membrane was blocked with BSA (bovine serum albumin)beforehand. More particularly, the membrane was blocked with 3 w/v % ofBSA in TBS-T at room temperature for 30 minutes. The membrane wasreacted with avidin-peroxidase (Vector) (50,000-fold diluted with TBS-T)at room temperature for 10 minutes. The membrane was washed with 50 mLof TBS-T three times. Then the membrane was reacted with ECL-plus(Amersham) for 5 minutes. A solution of ECL-plus was prepared accordingto the manufacturer's instructions. The reaction was stopped by washingthe membrane with 200 mL of water. Bands of fluorescence were visualizedby Molecular Imager (Bio-Rad) and quantified.

Exemplary Method 2 (Determination of Activated CDK1: Using Histone H1 asa Substrate and Utilizing FITC-Labeling)

This method was carried out in the same manner as Exemplary Method 1except that avidin-FITC was used in place of avidin-peroxidase andECL-plus was not reacted with the membrane in the above fourth step.

Exemplary Method 3 (Determination of Activated CDK2: Using Histone H1 asa Substrate and Utilizing FITC-Labeling)

First Step:

Tissue having a wet weight of 10 mg to 50 mg was put in a Eppendorf tube(1.5 mL volume), to which 800 μL of the lysis buffer mentioned in thefirst step of Exemplary Method 1 were added. The mixture was ground downwith a pestle. A basic movement of the pestle turning right to left at90° at a pressing force of 5 kg was repeated 10 times. The resultingcrude liquid of solubilized cells was passed through a syringe (1 mLvolume) fed with glass wool (about 0.1 g weight) and provided with adisk filter (Milipore) having a pore size of 0.45 μm at the tip thereof.Thereby was prepared a liquid of solubilized cells from which insolublesand lipid were removed. The total amount of protein contained in thesupernatant was measured by a DC protein kit (Bio-Rad) using bovine IgGas reference.

Second Step:

The Second Step was carried out in the same as in the second step ofExemplary Method 1.

Third Step:

To the suspension, 10 μL of a histone H1 solution (0.1 mg/mL solution in50 mM Tris-HCl, pH 7.4) were added. Then 10 μL of an ATP-γ S solution(10 mM aqueous solution) were added to the suspension. The resultingsuspension was incubated at 37° C. for 90 minutes with continuousoscillation. The beads were precipitated by centrifugation at 1,000 rpmfor 10 seconds and 30 μL of supernatant were collected. To 18 μL of thesupernatant, were added 15 μL of a binding buffer containing 150 mMTris-HCl, pH 9.2 and 5 mM EDTA. To the resulting supernatant, 10 μL of a5 mM iodoacetylfluorecein (Pierce) solution (in a 50 mM phosphate bufferof pH 6.0 and 50% dimethylsulfoxide) were added. The mixture wasincubated in a dark place at room temperature for 90 minutes. Thereaction of iodoacetamidofluorecein was stopped by adding 43 μL of β-ME.The reaction liquid was diluted 5 fold to 10 fold with TBS (50 mMTris-HCl, pH 7.4, 150 mM sodium chloride).

Fourth Step:

The diluted reaction liquid, 50 μL, was placed and absorbed onto a PVDFmembrane using a slot blotter. The membrane was washed with 50 mM TBS-T(a TBS solution containing 0.05 w/v % Tween 20) for 10 minutes threetimes with oscillation. Thereafter the membrane was washed with 200 mLof water and dried. Bands of fluorescence were visualized by MolecularImager (Bio-Rad) and quantified by an image analyzer.

Exemplary Method 4 (Determination of Activated CDK2: Using Histone H1 asa Substrate and Utilizing FITC-Labeling)

First Step:

This step was carried out in a manner similar to the first step ofExemplary method 1 using K562 cell line.

Second Step:

Samples were prepared in graded concentrations of 0, 25, 50, 100 and 200μg/mL of the total amount of protein of solubilized K562 cells in 500 Lof the lysis buffer and put in Eppendorf tubes. To each of the samples,10 μL of polyclonal anti-CDK antibody (200 μg/mL, Santa CruzBiotechnology) were added. To the resulting samples, a 1:1 (sepharosebeads:lysis buffer) slurry of 40 μL sepharose beads coated with ProteinA was added. The samples were incubated at 4° C. for an hour withcontinuous rotation. The beads were taken out of the samples and washedwith 1 mL of the lysis buffer twice. Then the beads were washed oncewith 100 mM Tris-HCl of pH 7.4 and 100 mM sodium chloride and furtherwith 100 mM Tris-HCl of pH 7.4.

Third Step:

To the beads, 50 μL of kinase buffer solution containing histone H1 (40mM Tris-HCl, pH 7.4, 18 mM magnesium chloride, 2 mM ATP-γ S, 6 μg/testhistone H1) were added. The resulting suspension was incubated at 37° C.for 90 minutes with continuous oscillation. The beads were precipitatedby centrifugation at 1,000 rpm for 10 seconds and 36 μL of supernatantwere collected. To 36 μL of the supernatant, were added 30 μL of abinding buffer containing 150 mM Tris-HCl and 2.5 mM EDTA of pH 9.2.Further, 20 μL of a 35 mM PEO-iodoacetylbiotin (Pierce) solution (in a50 mM phosphate buffer of pH 6.0) were added. The resulting mixture wasincubated in a dark place at room temperature for 90 minutes. Thereaction was stopped by adding an equivalent amount (86 μL) of β-ME. Thereaction liquid was diluted 5 fold to 10 fold with TBS (50 mM Tris-HCl,pH 7.4, 150 mM sodium chloride).

Fourth Step:

The diluted reaction liquid, 50 μL, was placed and absorbed onto a PVDFmembrane using a slot blotter. The obtained membrane was blocked with 2w/v % BSA for 30 minutes and washed with TBS for 5 minutes. Reaction wasconducted in a solution of avidin-FITC (Pierce) (500-fold diluted withTBS) at 37° C. for 60 minutes. After the reaction, the membrane waswashed with TBS three times and with water once, and dried. Bands offluorescence were visualized by Molecular Imager (Bio-Rad) and measured.

Production Example 1 Production of a Recombinant Vector Coding for a Rb(Retinoblastoma Protein) whose Cysteine Residue is Substituted byAlanine Residue and a Protein Produced by Expression of the Vector

(1) Construction of Expression Vector

First a cDNA coding human Rb was cloned from a cDNA library (Stratagene)of human placenta.

For constructing a plasmid for expressing a C-terminal (from Leu 769 toLys 928) of human Rb whose Cys 853 was uniquely modified to Ala, atwo-stage PCR was carried out using oligonucleotide primer with pJ3 Ωvector containing the full length of cDNA of human Rb.

1. First PCR

First, for amplifying a region of human Rb protein corresponding toLeu769 to Asp921 and substituting Cys 853 by Ala at the same time, thetwo-stage PCR was carried out using four species of primers. The primersused were primer Rb-1 (5′-ACA GGA TCC TTG CAG TAT GCT TCC-3′, into whicha BamHI site (as underlined) was introduced) and Rb-7 (5′-GCG AAT TCAATC CAT GCT ATC ATT-3′, into which a EcoRI site (as underlined) wasintroduced), which were primers at both ends, a primer Rb-2 whose 853position was changed into Ala codon (AGC) (5′-GCT GTT AGC TAC CAT CTGATT TAT-3′, the point modified codon is shown as underlined) and itscomplementary primer Rb-3 (5′-ATG GTA GCT AAC AGC GAC CGT GTG-3′). ThePCR was carried out with a primer set of Rb-1/Rb-2 and a primer set ofRb-3/Rb-7 using the total length of cDNA of human Rb as a template underthe following reaction conditions, to obtain PCR fragments 1 and 2,which were complementary in regions corresponding to the primer Rb-2 andthe primer Rb-3. Composition of Reaction Liquid (for fragment 1corresponding to nucleotide 2305 to 2565) PJ3 Ω-Rb 250 ng Taq DNApolymerase (TaKaRa Ex Taq, Takara Shuzo) 0.03U Buffer for TaKaRa Ex Taq(Takara Shuzo) MgCl₂ (Takara Shuzo) 2 mM dNTPs (Takara Shuzo) 250 μMPrimer Rb-1 1 μM Primer Rb-2 1 μM Total 50 μL

Composition of Reaction Liquid (for fragment 2 corresponding tonucleotide 2551 to 2763) PJ3 Ω-Rb 250 ng Taq DNA polymerase (TaKaRa ExTaq, Takara Shuzo) 0.03U Buffer for TaKaRa Ex Taq (Takara Shuzo) MgCl₂(Takara Shuzo) 2 mM dNTPs (Takara Shuzo) 250 μM Primer Rb-3 1 μM PrimerRb-7 1 μM Total 50 μLReaction Temperature

-   At 95° C. for 5 minutes-   At 94° C. for 30 seconds, at 55° C. for 1 minutes, at 72° C. for 1    minute (15 cycles)-   At 72° C. for 2 minutes    2. Treatment of Overhang

A-3′ overhangs of the PCR products were treated with Klenow under thefollowing conditions. Composition of Reaction Liquids PCR fragment 1 and2 Klenow fragment (Takara Shuzo) 0.07U Buffer for Klenow fragment(Takara Shuzo) dNTPs (Takara Shuzo) 250 μMReaction Temperature

-   At 37° C. for 1 hour    3. Second PCR

Thereafter, using a mixture of the PCR products as a template, PCR wascarried out with a primer set of Rb-1/Rb7 at both ends under thefollowing reaction conditions, to amplify a DNA fragment of 470 bpcorresponding to Leu769 to Asp921 in which Cys 853 was substituted byAla. Composition of Reaction Liquid Klenow-treated PCR fragment 1 and 2Taq DNA polymerase (TaKaRa Ex Taq, Takara Shuzo) 0.03U Buffer for TaKaRaEx Taq (Takara Shuzo) MgCl₂ (Takara Shuzo) 2 mM dNTPs (Takara Shuzo) 250μM Primer Rb-1 1 μM Primer Rb-7 1 μM Total 50 μLReaction Temperature

-   At 95° C. for 5 minutes-   At 94° C. for 30 seconds, at 55° C. for 1 minute, at 72° C. for 1    minute (15 cycles)-   At 72° C. for 2 minutes    4. Cloning into pMe1BacA

In order to express a Rb protein having a section signal added at the Nterminal, after the DNA fragment of 470 bp amplified in the previousstep 3 was digested with BamHI and EcoRI, the digested fragment wasinserted at the BamHI site and at the EcoRI site of pMe1BacA(Invitrogen). The obtained plasmid was referred to as pMe1BacA-Rb.

5. PCR

Using the obtained plasmid as a template, PCR was carried out usingprimer Rb-9 (5′-GCG AAT TCA TGA AAT TCT TAG TCA-3′, into which theEcorRI site was introduced as underlined) and primer Rb-5 (5′-GTT CTCGAG TCA ATC CAT GCT ATC ATT-3′, into which the XhoI site was introducedas underlined) under the following conditions, to amplify the DNAfragment of 540 bp to which the secretion signal was added. Compositionof Reaction Liquid pMelBacA-Rb 250 ng Taq DNA polymerase (TaKaRa Ex Taq,Takara Shuzo) 0.03U Buffer for TaKaRa Ex Taq (Takara Shuzo) MgCl₂(Takara Shuzo) 2 mM dNTPs (Takara Shuzo) 250 μM Primer Rb-9 1 μM PrimerRb-5 1 μM Total 50 μLReaction Temperature

-   At 94° C. for 5 minutes-   At 94° C. for 30 seconds, at 55° C. for 1 minutes, at 72° C. for 1    minute (25 cycles)-   At 72° C. for 2 minutes    6. Cloning into pFastBac1

The DNA fragment of 540 bp containing the secretion signal amplified inthe previous step 5 was digested with EcoRI and XhoI, and then wasinserted at the EcoRI site and at the XhoI site of pFastBac1 (Lifetech).

(2) Isolation of Recombinant Virus by Bac-To-Bac Baculovirus ExpressionSystem (Lifetech)

The expression plasmid obtained in the previous step 6 was used forisolating a recombinant virus, that is, according to the manufacturer'sinstructed protocol.

(3) Expression

The liquid of the recombinant virus prepared in the previous step (2)was infected to insect cells (High Five Cell, Invitroge) under thecondition of MOI=10 to express a region corresponding to Leu 769 to Asp921 containing Cys 853 substituted by Ala. The secretion of theexpressed protein into a medium (CELL405, JRH Biosciences) was confirmedby Western blotting using an anti-human Rb polyclonal antibody(Rb(C-15), Santa Cruz), and the medium was harvested five days after theinfection.

(4) Purification of Expressed Protein

The medium containing the Rb recombinant protein obtained in theprevious step (3) was exchanged with 50 mM MES buffer (pH 6.0) by PD-10column (Pharmacia), and then the protein is eluted by CM-5pW column(Tosoh) using a linear gradient of 0 to 1 M NaCl. The elution of the Rbrecombinant protein at about 0.3 M NaCl was confirmed by Westernblotting using an anti-human Rb polyclonal antibody (Rb(C-15), SantaCruz).

The obtained protein was used in place of histone H1 as a substrate inthe third step of Exemplary Method 1 for determination of CDK 4 or CDK6.

Exemplary Method 5 (Determination of Activated CDK4: Utilizing theRecombinant Human Rb Produced in Production Example 1 as a Substrate andFITC-Labeling

First Step:

This step was carried out in the same manner as in the first step ofExemplary Method 3.

Second Step:

Samples were prepared by placing the solubilized K562 cells and thelysis buffer into Eppendorf tubes of 1.5 mL volume so that 0, 50, 100,125 and 250 μg of the total amount of protein were in 500 μL of thelysis buffer. To each of the samples, 10 μL of a polyclonal anti-CDK 4antibody (200 μg/mL Santa Cruz Biotechnology) were added. A 1:1(sepharose beads:lysis buffer) slurry of 40 μL sepharose beads coatedwith Protein A was added to the resulting samples, which were incubatedat 4° C. for an hour with continuous rotation. The beads were taken outof the samples and washed with 1 mL of the lysis buffer twice. Then thebeads were washed once with 100 mM Tris-HCl of pH 7.4 and 100 mM sodiumchloride and further washed once with 100 mM Tris-HCl of pH 7.4.

Third Step:

To the obtained beads, 50 μL of phosphorylation solution containing therecombinant human Rb protein produced in Production Example 1 (40 mMTris-HCl, pH 7.4, 200 mM magnesium chloride, 3.3 mM ATP-γ S, 20 μLrecombinant human Rb protein solution: 50 mM MES buffer, pH 6.0) wereadded. The resulting suspension was incubated at 37° C. for 90 minuteswith continuous oscillation. The beads were precipitated bycentrifugation at 1,000 rpm for 10 seconds and 36 μL of supernatant werecollected. To 36 μL of the supernatant, were added 30 μL of a bindingbuffer containing 150 mM Tris-HCl and 2.5 mM EDTA of pH 9.2. To theresulting supernatant, 20 μL of a 50 mM PEO-iodoacetylbiotin (Pierce)solution (in a 50 mM phosphate buffer, pH 6.0). The supernatant wasincubated in a dark place at room temperature for 90 minutes.Thereafter, the supernatant was treated with an equivalent amount (86μL) of an SDS-sample loading buffer (0.125 M tris-HCl, pH 6.8, 4% SDS,10% β-ME, 25% glycerin, bromophenol blue) at 100° C. for 5 minutes.

Fourth Step:

The samples prepared in the third step was subjected to SDS-PAGE underthe condition of 20 μL/lane (pre-cast gel, 4-20% gradient, 10 mm×10 mm,Daiichi Kagaku Yakuhin). The conditions of the SDS-PAGE were incompliance with the instruction protocol of Daiichi Kagaku Yakuhin (60ma, 40 minutes). After the SDS-PAGE, the protein extended in the gel waselectrically transferred to PVDF membrane (10 V, 30 minutes, Westernblotting). The obtained membrane was blocked with 4 w/v % BSA for 30minutes and washed with TBS-T for 5 minutes. Subsequently, the membranewas reacted in a solution of avidin FITC (Pierce) (diluted 1,000 foldwith TBS-T) at 37° C. for 30 minutes. After reaction, the membrane waswashed with TBT-T twice and with water once, and dried. Bands offluorescence were visualized by Molecular Imager (Bio-Rad) and measured.

Determination of Activity of Activated CDK1

1. Production of Reference Curve Using Biotinyted Actin (BA) asReference Substance

Samples of BA are prepared in graded concentrations from 0 to 1000 ng/mLand each of the samples, 50 mL, is put into a slot. The sample in theslot is treated as in the fourth step of Exemplary Method 1. Since BA islabeled with peroxidase, BA produces a fluorophore from ECL-plus(fluorescent substrate) in the fourth step. The amount of fluorescenceis measured for the fluorophores generated in the samples containing thegraded concentrations of BA and is indicated by count (CNT) values. Theobtained data are plotted with the BA concentration in abscissa and theCNT value in ordinate, and a reference curve is produced under thereaction conditions of Exemplary Method 1. FIG. 1 shows the obtainedreference curve.

In the same manner, the samples in the slots are treated as in thefourth step of Exemplary Method 1, and a reference curve is produced inthe above-described manner under the reaction conditions of ExemplaryMethod 2. FIG. 2 shows the obtained reference curve.

2. Calculation of Activity of Activated CDK1

With regard to Exemplary Methods 1 and 2, blank samples are preparedwhich are treated according to Exemplary Method 1 except that theanti-CDK antibody is not added. The amount of fluorescence from theblank samples is measured. The obtained CNT values are converted to BAconcentrations using the reference curves produced for Exemplary MethodsA and B as described above. The activity of the activated CDK iscalculated by substituting the obtained BA concentrations into thefollowing formula:{(the converted BA concentration of a sample)−(the converted BAconcentration of the blank sample}×(the dilution ratio of thesample)=(the activity of the activated CDK1)  Formula 1

If the produced reference curve is not a sigmoid curve but a linear line(e.g., Exemplary Method 2), the activity of the activated CDK may becalculated by calculating (the CNT value of a sample)−(the CNT value ofthe blank sample), converting the obtained remainder to BA concentrationusing the reference curve and substituting the converted BAconcentration into the following formula:(the converted BA concentration of the remainder of the CNT value of thesample minus the CNT value of the blank sample)×(the dilution ration ofthe sample)=(the activity of the activated CDK)  Formula 2

Example 1 Measurement of Activity of Activated CDK1 of Sample TreatedAccording to Exemplary Method 1

The amount of fluorescent of 450-fold diluted samples prepared byExemplary Method 1 using the anti-CDK1 antibody was measured asdescribed above. Sample 1 was prepared using HeLa cells in a growthphase and Sample 2 was prepared using HeLa cells in a stationary phase.Since the reference curve was a sigmoid curve as shown in FIG. 1, theactivity of the activated CDK1 was calculated using Formula 1, that is,by producing blank samples as described above, measuring the amount offluorescence of the blank samples and substituting the CNT values of theblank samples and the samples into Formula 1. The results are shown inTable 2. TABLE 2 Activity of activated CDK1 CNT value (ng/slot) Sample 1147.6 6921.4  Blank of Sample 1 68.7 — Sample 2 133.3 5121.35 Blank ofSample 2 69.1 —

Example 2 Determination of Activity of Activated CDK1 of Sample TreatedAccording to Exemplary Method 2

The amount of fluorescent of 100-fold diluted samples prepared byExemplary Method 2 using the anti-CDK1 antibody was measured asdescribed above. Since the reference curve was linear as shown in FIG.2, it was found that the activity of the activated CDK1 was able to becalculated using any one of Formula 1 and Formula 2.

Determination of Activity of Activated CDK2

Example 3 Determination of Activity of Activated CDK2 of Samples Treatedby Exemplary Method 3

Sample 3 was prepared using the anti-CDK2 antibody according toExemplary Method 3. As controls, Sample 4 was prepared according toExemplary Method 3 without using the anti-CDK2 antibody, and Sample 5was prepared according to Exemplary Method 3 except that a non-specificIgG antibody was used instead of the anti-CDK2 antibody in the secondstep. The fluorescence bands of Samples 3 to 5 are shown in FIG. 3. Theamount of fluorescence of the bands were numerically represented byMolecular Imager (Bio-Rad). The obtained amount of fluorescence is shownin Table 3. TABLE 3 CNT value Sample 3 363 Sample 4 198 Sample 5 192

The above results show that a non-specific reaction was not observed inthe case where the non-specific IgG antibody was added instead of theanti-CDK2 antibody (Sample 5) and therefore that the activity of theactivated CDK2 was able to be determined by the method of the presentinvention.

Example 4 Determination of Activity of Activated CDK2 of Sample Treatedby Exemplary Method 4

1. Production of Reference Curve using K562 Cell Line (PremyelocyticLeukemia) as Reference Substance

The amount of fluorescence was measured about samples having gradedconcentrations of 0 to 200 μg/mL produced according to Exemplary Method4 and represented by CNT values. The obtained data were plotted with theconcentration of K562 cell line in abscissa and the CNT value inordinate to produce a reference curve. The obtained reference curve isshown in FIG. 4.

2. Curve of Activity of Activated CDK2

A specimen was prepared according to Exemplary Method 4 except that anunknown sample was used instead of the sample of solubilized K562 cells.The amount of fluorescence of the specimen was measured, and theobtained CNT value was converted to a CDK2 activity of the solubilizedK562 cells using the above-produced reference curve to calculate theactivity.

Determination of Activity of Activated CDK4

Example 5 Determination of Activity of Activated CDK4 Samples Treated byExemplary Method 5

Sample 6 was prepared according to Exemplary Method 5 using theanti-CDK4 antibody. As a control, Sample 7 was prepared according toExemplary Method 5 except that the anti-CDK4 antibody was not used. Thebands of fluorescence of Samples 6 and 7 are shown in FIG. 5.

Example to Prove the Specificity of Activated CDK2 Measurements toActivity of CDK2 using CDK-specific Inhibitor

Example 6 In the Case of Activated CDK2

First Step:

This step was carried out in the same manner as in the first step of theExemplary Method 1 using K562 cell line (premyelocytic leukemia).

Second Step:

A sample was prepared by placing the solubilized K562 cells and thelysis buffer into an Eppendorf tube of 1.5 mL volume so that 250 μg ofthe total amount of protein of the solubilized K562 cells were in 500 μLof the lysis buffer. To the sample, 10 μL of a polyclonal anti-CDK2antibody (200 μg/mL, Santa Cruz Biotechnology) were added. A 1:1(sepharose beads:lysis buffer) slurry of 40 μL sepharose beads coatedwith Protein A was added to the resulting sample, which was incubated at4° C. for an hour with continuous rotation. The beads were taken out ofthe sample and washed with 1 mL of the lysis buffer twice. Then thebeads were washed once with 100 mM Tris-HCl of pH 7.4 and 100 mM sodiumchloride and further washed once with 100 mM Tris-HCl of pH 7.4

Third Step:

As CDK inhibitors, were used Butyrolactone 1 (Calbiochem) which wereinhibitors to CDK1 and CDK2 and Staurosporine (Calbiochem) which had abroad inhibition spectrum including inhibition to CDK2. Phosphorylationsolutions (40 mM Tris-HCl, pH 7.4, 18 mM magnesium chloride, 2 mM ATP-γS, 6 μg/test histone H1), 50 μL, containing histone H1 and the CDKinhibitors (final concentrations of 0, 1, 10, 30, 100 μM ofButyrolactone, or final concentrations of 0, 0.3, 1, 10, 30 μM ofStaurosporine) were added to the beads. The resulting suspensions wereincubated at 37° C. for 90 minutes with continuous oscillation. Thebeads were precipitated by centrifugation at 1,000 rpm for 10 seconds,and 36 μL of supernatant were collected. To 36 μL of the supernatant, 30μL of a binding buffer containing 150 mM Tris-HCl and 5 mM of EDTA of pH9.2 was added. Further, 20 μL of 50 mM PEO-iodoacetylbiotin (Pierce)solution (50 mM phosphate buffer, pH 6.0) was added and the resultingmixture was incubated in a dark place at room temperature for 90minutes. Thereafter, the mixture was treated with an equivalent amount(86 μL) of an SDS-sample loading buffer (0.125 M tris-HCl, pH 6.8, 4%SDS, 10% β-ME, 25% glycerine, bromophenol blue) at 100° C. for 5minutes.

Fourth Step:

The samples prepared in the third step was subjected to SDS-PAGE underthe condition of 20 μL/lane (pre-cast gel, 4-20% gradient, 10 mm×10 mm,Daiichi Kagaku Yakuhin). The conditions of the SDS-PAGE were incompliance with the instructions of Daiichi Kagaku Yakuhin (60 mA, 40minutes). After the SDS-PAGE, the protein extended in the gel waselectrically transferred to a PVDF membrane (10V, 30 minutes, Westernblotting). The obtained membrane was blocked with a 4 w/v % BSA for 30minutes and washed with TBS-T for 5 minutes. Subsequently, the membranewas reacted in a solution of avidin FITC (Pierce) (diluted 1,000 foldwith TBS-T) at 37° C. for 30 minutes. After reaction, the membrane waswashed with TBS-T twice and with water once, and dried. Bands offluorescence were visualized by Molecular Imager (Bio-Rad) and measured.

The visualized bands of fluorescence were numerically and graphicallyrepresented by an image analyzer. The results are shown in FIG. 6,wherein none represents the result of the above step without addition ofthe CDK inhibitors, DMSO represents the result of the above step withaddition only of a solvent (dimethylsulfoxide) for the CDK inhibitorsand IP (-) represents the result of the above step without addition ofthe CDK2 antibody. As shown in FIG. 6, the activity of CDK2 wasinhibited dependently upon the amounts of the inhibitors. The resultsshows that the determined activity was specific to CDK2.

As shown above, the method of the present invention can measure theactivity of cell cycle controlling factors accurately without usingradioisotopes.

1. A set of reagents for measuring the activity of cyclin-dependentkinase in a sample prepared from a living cell, which comprises: ananti-cyclin-dependent kinase antibody, a substrate for cyclin-dependentkinase, adenosine 5′-O-(3-thiotriphosphate) (ATP-γS); and a labelingfluorophore or a labeling enzyme for coupling with a reaction product ofthe substrate and the ATP-γS.
 2. The set of reagents according to claim1, wherein the cyclin-dependent kinase is selected from the groupconsisting of CDK1, CDK2, CDK4 and CDK6.
 3. The set of reagentsaccording to claim 1, further comprising a lysis buffer for solubilizingthe living cell.
 4. The set of reagents according to claim 3, whereinthe lysis buffer contains a surfactant, a protease inhibitor and aphosphatase inhibitor.
 5. The set of reagents according to claim 1,wherein the cyclin-dependent kinase is CDK1 or CDK2 and the substrate ishistone H1.
 6. The set of reagents according to claim 1, wherein thecyclin-dependent kinase is CDK4 or CDK6 and the substrate is Rb whosecysteine residue is substituted by an amino acid residue which does notcontain thiol residue.
 7. The set of reagents according to claim 1,wherein the labeling fluorophore is a fluorescent dye.
 8. The set ofreagents according to claim 7, wherein the fluorescent dye is FITC. 9.The set of reagents according to claim 1, wherein the labeling enzyme isperoxidase.
 10. The set of reagents according to claim 1, furthercomprising a coupling reaction stopping agent for stopping a couplingreaction between the labeling fluorophore or the labeling enzyme and thereaction product.
 11. The set of reagents according to claim 10, whereinthe coupling reaction stopping agent comprises a thiol.
 12. Acombination of reagents for measuring activity of cyclin-dependentkinase in a sample prepared from a living cell, which comprises: ananti-cyclin-dependent kinase antibody, a substrate for cyclin-dependentkinase, adenosine 5′-O-(3-thiotriphosphate) (ATP-γS); and a labelingfluorophone or a labeling enzyme for coupling with a reaction product ofthe substrate and the ATP-γS.
 13. The combination of reagents accordingto claim 12, further comprising a lysis buffer for solubilizing theliving cell.
 14. The combination of reagents according to claim 13,wherein the lysis buffer contains a surfactant, a protease inhibitor anda phosphatase inhibitor.
 15. The combination of reagents according toclaim 12, wherein the further comprising a coupling reaction stoppingagent for stopping a coupling reaction between the labeling fluorophoreor the labeling enzyme and the reaction product.
 16. The combination ofreagents according to claim 15, wherein the coupling reaction stoppingagent comprises a thiol.